Target populations of oligodendrocyte precursor cells and methods of making and using same

ABSTRACT

This application provides for enriched target populations oligodendrocyte precursor cells (OPCs) that can differentiate into oligodendrocytes. The target OPCs may be expanded and optionally subjected to conditions to induce their differentiation into oligodendrocytes. The target OPCs and their progeny are useful for the treatment of disease associated with demyelination of central nervous system axons.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/140,410, filed Dec. 23, 2008.

FIELD OF THE INVENTION

This invention relates to isolation, characterization, proliferation,differentiation and transplantation of a population of oligodendrocyteprecursor cells.

BACKGROUND

During development of the central nervous system (“CNS”), multipotentneural precursor cells, also known as neural stem cells, proliferate andgive rise to transiently dividing progenitor cells that eventuallydifferentiate into the cell types that compose the adult brain. Stemcells (from other tissues) have classically been defined as having theability to self-renew (i.e., form more stem cells), to proliferate, andto differentiate into different phenotypic lineages. In the case ofneural stem cells, this includes neurons, astrocytes andoligodendrocytes. Neural stem cells have been isolated from severalmammalian species, including mice, rats, pigs and humans. See, e.g., WO93/01275, WO 94/09119, WO 94/10292, WO 94/16718 and Cattaneo et al.,Mol. Brain. Res., 42, pp. 161-66 (1996), the disclosures of which areherein incorporated by reference in their entireties. The main functionof oligodendrocytes is the myelination of axons in the central nervoussystem of higher vertebrates. Oligodendrocyte precursor cells (OPCs)precede oligodendrocytes.

SUMMARY

The invention provides for enriched target populations ofoligodendrocyte precursor cells (OPCs) that can further differentiateinto oligodendrocytes. According to some embodiments, populations ofOPCs are provided that are substantially enriched for cells expressingthe PDGFRα antigen.

According to some embodiments, the target OPCs are PDGFRα⁺ andadditionally CD105⁻. According to some embodiments, the targetpopulations of cells are enriched for OPCs that are immunopositive forPDGFRα (PDGFRα⁺) and immunonegative for CD105 (CD105⁻). According tosome embodiments, the target populations of cells are enriched for OPCsthat are immunopositive for PDGFRα (PDGFRα⁺), immunopositive for CD133(CD133⁺), and immunonegative for CD105 (CD105⁻).

According to some embodiments, the target OPCs are PDGFRα⁺ andadditionally A2B5^(lo), A2B5⁻, or mixture thereof (A2B5^(lo/−)).According to some embodiments, the target populations of cells areenriched for OPCs that are immunopositive for PDGFRα (PDGFRα⁺),immunopositive for CD133 (CD133⁺), and immunonegative for A2B5 (A2B5⁻).According to some embodiments, the target population of OPCs arePDGFRα⁺, A2B5^(lo/−). According to some other embodiments, the targetpopulation of OPCs are PDGFRα⁺, CD133⁺, A2B5⁻. According to some otherembodiments, the target population of OPCs are PDGFRα⁺, CD133⁺,A2B5^(lo/−). According to some other embodiments, the target populationof OPCs are PDGFRα⁺, CD133⁺, A2B5⁻, PSA-NCAM⁻. According to some otherembodiments, the target population of OPCs are PDGFRα⁺, CD133⁺,A2B5^(lo/−), PSA-NCAM⁻. According to some other embodiments, the targetpopulation of OPCs are PDGFRα⁺, CD133⁺, A2B5⁻, PSA-NCAM^(lo/−).According to some other embodiments, the target population of OPCs arePDGFRα⁺, CD133⁺, A2B5^(lo/−), PSA-NCAM^(lo/−).

According to some embodiments, the target OPCs are PDGFRα⁺, CD105⁻ andadditionally A2B5^(lo), A2B5⁻, or mixture thereof (A2B5^(lo/−)).According to some embodiments, the target populations of cells areenriched for OPCs that are immunopositive for PDGFRα (PDGFRα⁺),immunopositive for CD133 (CD133⁺), immunonegative for A2B5 (A2B5⁻), andimmunonegative for CD105 (CD105⁻). According to some embodiments, thetarget population of OPCs are PDGFRα⁺, CD105⁻, A2B5^(lo/−). According tosome other embodiments, the target population of OPCs are PDGFRα⁺,CD133⁺, CD105⁻, A2B5⁻. According to some other embodiments, the targetpopulation of OPCs are PDGFRα⁺, CD133⁺, CD105⁻, A2B5^(lo/−). Accordingto some other embodiments, the target population of OPCs are PDGFRα⁺,CD133⁺, CD105⁻, A2B5⁻, PSA-NCAM⁻. According to some other embodiments,the target population of OPCs are PDGFRα⁺, CD133⁺, CD105⁻, A2B5^(lo/−),PSA-NCAM⁻. According to some other embodiments, the target population ofOPCs are PDGFRα⁺, CD133⁺, CD105⁻, A2B5⁻, PSA-NCAM^(lo/−). According tosome other embodiments, the target population of OPCs are PDGFRα⁺,CD133⁺, CD105⁻, A2B5^(lo/−), PSA-NCAM^(lo/−).

According to some embodiments, methods of identifying, isolating, orenriching target populations of oligodendrocyte precursor cells, areachieved by contacting a population of cells containing at least one OPCwith a reagent that binds to the surface marker antigen expressed on thecell surface of an OPC. According to preferred embodiments, enrichedpopulations of target oligodendrocyte precursor cells are achieved bycontacting a population of cells containing at least one OPC with areagent that binds to PDGFRα. Preferably, the reagent is an antibodythat binds to PDGFRα. Use of traditional techniques for cell sorting,such as by immunoselection (e.g., FACS), permits identification,isolation, and/or enrichment for cells in which contact between thereagent and the PDGFRα antigen has been detected.

According to some embodiments, the invention provides methods forproducing populations enriched for target oligodendrocyte precursorcells by contacting neural or neural derived cells with a monoclonalantibody that binds to a negative selection marker that is not found onthe target oligodendrocyte precursor cells; selecting the cells thatbind to this monoclonal antibody; and removing the bound cells. Theremaining cells in the population are enriched for oligodendrocyteprecursor cells. Those skilled in the art will recognize that a negativeselection marker is a marker (i.e., an antigen) that is present only onnon-OPC cells. In various embodiments, the monoclonal antibody may befluorochrome conjugated or may be conjugated to magnetic particles, andthe selection may be by fluorescence activated cell sorting, highgradient magnetic selection, by attachment to and disattachment from thesolid phase, or any other commonly used selection technique. Inpreferred embodiments, the population containing neural orneural-derived cells is obtained from a suspension culture, an adherentculture, or from fresh neural tissue. These methods may also involve thestep of further enriching the population for oligodendrocyte precursorcells by contacting the remaining cells with a second antibody or seriesof antibodies. For example, target populations of OPCs may be enrichedby contacting the culture of neural or neural derived cells with anantibody that specifically binds to CD133 followed by contacting theremaining cells with an antibody that specifically binds PDGFRα toproduce populations enriched for oligodendrocyte precursor cells thatare immunopositive for both CD133 and PDGFRα. In addition the culture ofneural or neural derived cells may be contacted with an antibody thatspecifically binds PDGFRα to produce populations enriched for OPCs thatare immunopositive for PDGFRα.

According to some embodiments, the invention provides methods forisolating a oligodendrocyte precursor cell (OPC), by selecting from apopulation of neural or neural-derived cells for cells that areimmunopositive for CD133 (CD133⁺ cells); eliminating thenon-immunoreactive (CD133⁻) cells from the population; and selectingfrom the remaining population for at least one cell that isimmunopositive for PDGFRα (PDGFRα⁺), e.g., binds to monoclonal antibodyPDGFRα. In other embodiments, the invention provides methods forproducing a population enriched for oligodendrocyte precursor cells bycontacting neural or neural derived cells containing at least onemultipotent neural stem cell with an antibody that specifically binds toPDGFRα and selecting those cells that are PDGFRα^(hi), wherein theselected cells are enriched for oligodendrocyte precursor cells ascompared with the neural or neural derived cells. Those skilled in theart will recognize that the neural or neural derived cells can beobtained from a neurosphere culture (cell clusters or cell aggregates)or from an adherent culture. In some embodiments, this method alsoinvolves the step of eliminating those cells that are PDGFRα^(lo/med)from the population.

According to some embodiments, the invention provides methods forisolating a oligodendrocyte precursor cell (OPC), by selecting from apopulation of neural or neural-derived cells for cells that areimmunonegative for CD105 (CD105⁻ cells); eliminating the immunoreactive(CD105⁺) cells from the population; and selecting from the remainingpopulation for at least one cell that is immunopositive for PDGFRα(PDGFRα⁺), e.g., binds to monoclonal antibody PDGFRα. According to someembodiments, the invention provides methods for isolating aoligodendrocyte precursor cell (OPC), by selecting from a population ofneural or neural-derived cells for cells that are immunopositive forPDGFRα (PDGFRα⁺), e.g., binds to monoclonal antibody PDGFRα; eliminatingthe non-immunoreactive (PDGFRα⁻) cells from the population; andselecting from the remaining population for at least one cell that isimmunonegative for CD105 (CD105⁻).

In other embodiments, the invention provides methods for producing apopulation enriched for oligodendrocyte precursor cells by contactingneural or neural derived cells containing at least one multipotentneural stem cell with an antibody that specifically binds to PDGFRα andselecting those cells that are PDGFRα^(hi), wherein the selected cellsare enriched for oligodendrocyte precursor cells as compared with theneural or neural derived cells. Those skilled in the art will recognizethat the neural or neural derived cells can be obtained from aneurosphere culture or from an adherent culture. In some embodiments,this method also involves the step of eliminating those cells that arePDGFRα^(lo/med) from the population.

According to some embodiments, the invention provides methods forproducing a population enriched for oligodendrocyte precursor cells byeliminating cells that are positive for markers of differentiated cellsor fibroblasts cells from a population of neural or neural-derived cells(e.g., CD105). This may be accomplished by contacting the populationwith a monoclonal antibody directed to such markers of differentiatedcells and removing those cells that bind to this monoclonal antibody.This resulting population of cells may be further enriched using any ofthe methods described herein. By way of non-limiting example, the methodmay involve the step of further enriching the population foroligodendrocyte precursor cells by contacting the remaining cells withan antibody that specifically binds to PDGFRα. In various otherpreferred embodiments, the fraction may optionally be enriched byselecting from the remaining cells for cells are PDGFRα⁺, CD105⁻,CD133⁺, A2B5^(lo/−), PSA-NCAM^(lo/−) and mixtures and combinationsthereof.

According to additional embodiments, the invention provides methods forproliferating enriched populations of oligodendrocyte precursor cells byintroducing at least one selected cell to a serum-free culture mediumcontaining one or more growth factors selected from the group consistingof LIF, EGF, bFGF, PDGF-AA, PDGF-AB, PDGF-BB, Sonic hedgehog (Shh),IGF1, CTNF, Noggin, and NT3 and combinations thereof; and proliferatingat least one selected cell in the culture medium. Preferably, the methodfor proliferating enriched populations of oligodendrocyte precursorcells comprises introducing at least one selected cell to a serum-freeculture medium containing one or more growth factors selected from thegroup consisting of PDGF-AA, NT3, bFGF, IGF1 and combinations thereof;and proliferating at least one selected cell in the culture medium.

Also provided are methods of proliferating and differentiating targetOPCs. According to some embodiments, the induction of proliferation (anddifferentiation) of the OPCs can be done either by culturing the cellsin suspension or on a substrate onto which they can adhere.Alternatively, proliferation and differentiation of OPCs can be induced,under appropriate conditions, in the host in the following combinations:(1) proliferation and differentiation in vitro, then transplantation,(2) proliferation in vitro, transplantation, then further proliferationand differentiation in vivo, (3) proliferation in vitro, transplantationand differentiation in vivo, and (4) proliferation and differentiationin vivo. Proliferation and differentiation in vivo or in situ caninvolve a non-surgical approach that coaxes OPCs to proliferate in vivowith pharmaceutical manipulation.

According to some embodiments, methods are provided for treating orameliorating a demyelinating or dysmyelinating disease or disorder in amammal, comprising administering to the mammal a target OPC or apopulation of target OPCs.

The mammal preferably harbors a demyelinating or dysmyelinating disease,including, but not limited to, multiple sclerosis, acute disseminatedencephalomyelitis, diffuse cerebral sclerosis, necrotizing hemorrhagicencephalitis, radiation induced myelination disorders, transversemyelitits, Pelizaeus-Merzbacher disease (PMD), Cerebral palsy (CP), andleukodystrophies. The disease is preferably multiple sclerosis,Pelizaeus-Merzbacher disease, or Cerebral palsy. The mammal mayadditionally receive at least one biological agent that is capable ofincreasing the number of OPCs and/or at least one factor that is knownto stimulate oligodendrocyte differentiation, growth, proliferation, orsurvival. The OPCs, oligodendrocyte promoting factor(s), biologicalagent(s), and/or other factor(s) may be administered in any manner thatresults in contact of the factor and or agent with target OPCs in themammal, such as systemically (e.g., subcutaneously) or in situ.Preferably, the OPCs, oligodendrocyte promoting factor(s), biologicalagent(s), and/or other factor(s) may be administered into the brain,more preferably into the lateral ventricle of the brain or into thebrain parenchyma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sagital section of shiverer/scid mouse counter stained withmethyl green to highlight cell nuclei. Arrows indicate the 3 areas ofthe brain injected with human OPCs. Abbreviations: cc, corpus callosum;fb, fimbria; cb, cerebellum.

FIG. 2. Example of OPC engraftment in the neonatal shiverer/scid mouse.Top panel shows sc121 staining, highlighting all donor derived cells.The bottom panel shows MBP staining in a serial sister section. Dottedregions of interest have been drawn to indicate the areas of engraftmentand of corresponding myelination. In this example, the superiorcolliculus was targeted. Cell line information: FBr 2711, P6, 8 weekspost transplant. Abbreviations used: str, striatum; cc, corpus callosum;sc, superior colliculus.

FIG. 3. Example of MBP-GFP transduced OPC engraftment in the neonatalshiverer/scid mouse. Top panel shows GFP staining, highlighting donorderived cells that are actively transcribing the mbp gene. The bottompanel shows MBP staining in a serial sister section. Dotted regions ofinterest have been drawn around the cerebellum and are shown in thepanels on the right at higher magnification. Note that there is adigital misalignment of the cerebellum in the MBP panel as indicated bythe white arrows. Cell line information: FBr 2703, P6, 8 weeks posttransplant. Abbreviations used: cb, cerebellum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only not intended tobe limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed.

DEFINITIONS

For the purposes of promoting an understanding of the embodimentsdescribed herein, reference will be made to preferred embodiments andspecific language will be used to describe the same. The terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention.As used throughout this disclosure, the singular forms “a,” “an,” and“the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a cell” includes aplurality of such cells, and a reference to “an antibody” is a referenceto one or more antibodies, and so forth.

As used herein, the term “target cell population” denotes those cellswhich are desirably being purified or enriched. Preferably, the targetcell population are oligodendrocyte precursor cells that display thedistinctive pattern of cell markers as described herein.

“Oligodendrocytes” (OLs) or oligodendroglia are best known as themyelin-forming cells of the central nervous system (CNS). The term“oligodendrocyte precursor cell” or “OPC” refers to the immature form ofoligodendrocytes that are capable of differentiating into myelin formingcells of the CNS under certain conditions. This term includesoligodendrocyte precursor cells isolated from primary tissue and cellscultured in vitro into OPCs, as well as the progeny of sucholigodendrocyte precursor cells, and thus includes both OPCs anddaughter OPCs.

The term “neural stem cells” is the more general term used forundifferentiated, multipotent, self-renewing, neural cells. A neuralstem cell is a clonogenic multipotent stem cell which is able to divideand, under appropriate conditions, has self-renewal capability and caninclude in its progeny daughter cells which can terminally differentiateinto neurons, astrocytes, and oligodendrocytes. Hence, the neural stemcell is “multipotent” because stem cell progeny have multipledifferentiation pathways. A neural stem cell is capable of selfmaintenance, meaning that with each cell division, one daughter cellwill also be a stem cell.

The non-stem cell progeny of a neural stem cell are typically referredto as “progenitor” or “precursor” cells, which are capable of givingrise to various cell types within one or more lineages. The term “neuralprogenitor cell” or “neural precursor cell” refers to anundifferentiated cell derived from a neural stem cell and is not itselfa stem cell. Some progenitor cells can produce progeny that are capableof differentiating into more than one cell type. For example, an O-2Acell is a glial progenitor cell that gives rise to oligodendrocytes andtype II astrocytes, and, thus, could be termed a “bipotential”progenitor cell. A distinguishing feature of a progenitor cell is that,unlike a stem cell, it does not exhibit self maintenance. Moreover,progenitor cells are typically thought to be committed to a particularpath of differentiation and will, under appropriate conditions,eventually differentiate into glia or neurons.

The terms “neural cells” or “neural derived cells” refers broadly tocells associated with the central nervous system (CNS) of an organism,for example, neurons, glial cells, and precursor cells. As used herein,neural cells may be cells that are isolated or derived from neuraltissue, as well as any cell, regardless of origin, having at least anindication of neuronal or glial phenotype, such as staining for one ormore neuronal or glial markers or which will differentiate into cellsexhibiting neuronal or glial markers. Thus, the term may be used as ageneral term to refer to, for example, primary cells isolated andcultured in vitro; cultured immortalized cells derived from a neuraltissue, neural tissue cells; and/or cells cultured to express a neuralphenotype. The term is meant to be all-encompassing with respect tocells exhibiting a neural cell phenotype and/or isolated from neuraltissue. Thus, the term neural cells also includes cells which are neuralprecursor cells as well as differentiated neural cells. As used herein,the term “neuronal cells” refers to neurons.

The term “positive selection” refers to a process in which the targetcell population is purified or enriched by removing the target cellpopulation from a mixture of cell populations by directly binding thetarget cell population to reagents having affinity therefore.

In contrast, the term “negative selection” refers to a process in whichthe target cell population is purified or enriched by removing nontargetcell populations from the mixture of cells by binding the nontarget cellpopulations to reagents having affinity therefore. For example, CD45 isthe T200/leucocyte common antigen. Human central nervous system stemcells (CNS-SC), and preferably those that can initiate neurospheres, andcultures containing them, are additionally characterized as lackingcertain cell surface markers such as CD45. Thus, reagents that recognizeCD45 may be useful in a negative selection process to remove nontargetcells.

A “primary neurosphere” is a neurosphere generated by culturing braintissue. Typically, the brain tissue is dissected and mechanicallydissociated before being cultured in appropriate media and allowed toform neurospheres. Exemplary methods are described in, for instance,U.S. Pat. No. 5,750,376, the disclosure of which is incorporated hereinby reference in its entirety.

A “secondary neurosphere” is a neurosphere generated by dissociating(passaging) a primary neurosphere and culturing the dissociated cellsunder conditions which result in the formation of neurospheres fromsingle cells.

A “mammal” is any member in the mammalian family. A mammal is preferablya primate, rodent, feline, canine, domestic livestock (such as cattle,sheep, goats, horses, and pigs), and most preferably a human.

A “demyelinating disease” or a “dysmyelinating disorder” is a disease,disorder, or medical condition that is caused by or associated withinadequate amounts myelin. Demyelination is the process of myelinremoval i.e., loss of myelin that existed before. Dysmyelination occurswhere no or inadequate amounts of myelin forms, e.g., due todysfunctional OPCs or oligodendrocytes (OLs). The end result ofdemyelination and dysmyelination is hypomyelination. Examples of thesediseases, disorders, or conditions include, for example, multiplesclerosis (including the relapsing and chronic progressive forms ofmultiple sclerosis, acute multiple sclerosis, neuromyelitis optica(Devic's disease)), diffuse cerebral sclerosis (including Shilder'sencephalitis periaxialis diffusa and Balo's concentric sclerosis).Demyelinating diseases or dysmyelinating disorders also include avariety of diseases wherein demyelination is caused by viral infections,vaccines, spinal cord injury, and genetic disorders. Examples of thesedemyelinating diseases or dysmyelinating disorders include acutedisseminated encephalomyelitis (occurring after measles, chicken pox,rubella, influenza or mumps; or after rabies or small pox vaccination),necrotizing hemorrhagic encephalitis (including hemorrhagicleukoencephalitis), and leukodystrophies (including Krabbe's globboidleukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy,adrenomyeloneuropathy, adrenomyeloneuropathy, radiation inducedmyelination disorders, transverse myelitits, Pelizaeus-Merzbacherdisease (PMD), Canavan's disease and Alexander's disease). Thedemyelinating disease or dysmyelinating disorder is preferably multiplesclerosis, cerebral palsy, diffuse cerebral sclerosis, orPelizaeus-Merzbacher disease (PMD), and, most preferably,Pelizaeus-Merzbacher disease.

“Treating” or “ameliorating” means the reduction or complete removal ofthe symptoms of a disease or medical condition.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. The effective amount of a giventherapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the animalto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods in theart.

The term “ventricle” refers to any cavity or passageway within the CNSthrough which cerebral spinal fluid flows. Thus, the term not onlyencompasses the lateral, third, and fourth ventricles, but alsoencompasses the central canal, cerebral aqueduct, and other CNScavities.

Cell Markers

This invention provides for the identification, isolation, enrichment,and culture of oligodendrocyte precursor cells. The target cellpopulation of OPCs can be characterized by their expression of cellsurface markers. While it is commonplace in the art to refer to cells as“positive” or “negative” for a particular marker, actual expressionlevels are a quantitative trait. The number of molecules on the cellsurface can vary by several logs, yet still be characterized as“positive”. It is also understood by those of skill in the art that acell which is negative for staining, i.e., the level of binding of amarker specific reagent is not detectably different from a control, e.g.an isotype matched control; may express minor amounts of the marker.Characterization of the level of staining permits subtle distinctionsbetween cell populations.

The staining intensity of cells can be monitored by flow cytometry,where lasers detect the quantitative levels of fluorochrome (which isproportional to the amount of cell surface marker bound by specificreagents, e.g. antibodies). Flow cytometry, or FACS, can also be used toseparate cell populations based on the intensity of binding to aspecific reagent, as well as other parameters such as cell size andlight scatter. Although the absolute level of staining may differ with aparticular fluorochrome and reagent preparation, the data can benormalized to a control.

In order to normalize the distribution to a control, each cell isrecorded as a data point having a particular intensity of staining.These data points may be displayed according to a log scale, where theunit of measure is arbitrary staining intensity. In one example, thebrightest cells in a population are designated as 4 logs (i.e., 10,000times) more intense than the cells having the lowest level of staining.When displayed in this manner, it is clear that the cells falling in thehighest log of staining intensity are bright, while those in the lowestintensity are negative. The “low” staining cells, which fall in the 2-3log(i.e., 100-1000 fold) of staining intensity, may have properties thatare unique from the negative and positive cells. An alternative controlmay utilize a substrate having a defined density of marker on itssurface, for example a fabricated bead or cell line, which provides thepositive control for intensity. The “low” designation indicates that thelevel of staining is above the brightness of an isotype matched control,but is not as intense as the most brightly staining cells normally foundin the population.

For the purpose of defining staining intensity of a particular antibody,an isotype matched control will define the signal intensity of“non-specific” or “negative” staining. Whereas any staining whichresults in signal intensity above that of the control is considered tobe “positive” staining. The boundary demarcating negative and positivestaining is conventionally set such that the frequency of events to theleft of, or below, the boundary is >0.99 and <1.0. Positive stainingintensity can then be further subdivided and categorized as low, medium,or high by defining an arbitrary scale from the control boundary to thehighest recorded signal intensity and defining two additional lines ofdemarcation at the 33^(rd) and 66^(th) percentiles, respectively.Signals measured in the lower-third, middle-third, and upper-third ofthese defined groups can then be designated as low, medium, and highstaining intensity, respectively.

Cell Sorting

The use of cell surface antigens to isolate, select, or enrich for OPCcells provides a means for the positive and negative immunoselection oftarget OPC populations, as well as for the phenotypic analysis of targetOPC cell populations using flow cytometry. For the preparation ofsubstantially pure target OPC populations, a subset of OPCs is separatedfrom other cells on the basis of PDGFRα binding. OPCs may be furtherseparated by binding to other surface markers known in the art. Cellsselected for expression of PDGFRα antigen, for example, may be furtherpurified by the positive and negative immunoselection of other targetOPC markers as disclosed herein.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography and “panning”with antibody attached to a solid matrix, e.g. plate, or otherconvenient technique. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. Dead cellsmay be eliminated by selection with dyes associated with dead cells(propidium iodide [PI], LDS). Any technique may be employed which is notunduly detrimental to the viability of the selected cells.

Conveniently, the antibodies are conjugated with labels to allow forease of separation of the particular cell type, e.g. magnetic beads;biotin, which binds with high affinity to avidin or streptavidin;fluorochromes, which can be used with a fluorescence activated cellsorter; haptens; and the like. Multi-color analyses may be employed withthe FACS or in a combination of immunomagnetic separation and flowcytometry. Multi-color analysis is of interest for the separation ofcells based on multiple surface antigens, e.g. PDGFRα⁺, CD105⁻, CD133⁺,CD24⁻, etc. Fluorochromes which find use in a multi-color analysisinclude, for example, phycobiliproteins, e.g. phycoerythrin andallophycocyanins; fluorescein and Texas red. A negative designationindicates that the level of staining is at or below the brightness of anisotype matched negative control. A “dim”, “lo”, or “low” designationindicates that the level of staining may be near the level of a negativestain, but may also be brighter than an isotype matched control.

For example, the PDGFRα antibody is directly or indirectly conjugated toa magnetic reagent, such as a superparamagnetic microparticle(microparticle). Direct conjugation to a magnetic particle is achievedby use of various chemical linking groups, as known in the art. Antibodycan be coupled to the microparticles through side chain amino orsulfhydryl groups and heterofunctional cross-linking reagents. A largenumber of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyldithio) propionicacid N-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

Alternatively, the antibody is indirectly coupled to the magneticparticles. The antibody may be directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include, for examples digoxin, digoxigenin,FITC, dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods forconjugation of the hapten to a protein, i.e. are known in the art, andkits for such conjugations are commercially available.

Target OPC populations are selected by bringing neural or neural-derivedcells into contact with the antibody or reagent that binds the surfacemarker. For example, antibody is added to a cell sample. The amount ofantibody or other reagent necessary to bind a particular cell subset isempirically determined by performing a test separation and analysis. Forexample, the cells and antibody/reagent are incubated for a period oftime sufficient for complexes to form, preferably at least about 5 min,more preferably at least about 10 min, and usually not more than one hr,more usually not more than about 30 min. The cells may additionally beincubated with antibodies or binding molecules specific for cell surfacemarkers known to be present or absent on OPCs.

The labeled cells are separated in accordance with the specific antibodypreparation. Fluorochrome labeled antibodies are useful for FACSseparation, magnetic particles for immunomagnetic selection,particularly high gradient magnetic selection (HGMS), etc. Exemplarymagnetic separation devices are described in WO 90/07380,PCT/US96/00953, and EP 438,520, the disclosures of which are hereinincorporated by reference in their entireties.

The purified cell population may be collected in any appropriate medium.Various media are commercially available and may be used, includingDulbecco's Modified Eagle Medium (DMEM), Hank's Basic Salt Solution(HBSS), Dulbecco's phosphate buffered saline (DPBS), RPMI, Iscove'smodified Dulbecco's medium (IMDM), phosphate buffered saline (PBS) with5 mM EDTA, etc., frequently supplemented with fetal calf serum (FCS),bovine serum albumin (BSA), human serum albumin (HSA), etc.

Populations highly enriched for target OPCs are achieved in this manner.The desired cells will be 30% or more of the cell composition,preferably 50% or more (e.g., 60% or more, 70% or more, 75% or more, 80%or more, 85% or more, 90% or more, 95% or more) of the cell population,more preferably 90% or more (e.g., 92% or more, 94% or more, 96% ormore, or 98% or more) of the cell population, and most preferably 95% ormore (e.g., 97%, 99%) (substantially pure) of the cell population. Thedegree of enrichment obtained, and actually used, depends on a number offactors, including, but not limited to, the method of selection, themethod of growth, and/or the dose of the cells that are placed inculture.

Isolation, Enrichment, and Selection of Cells

The invention provides for the isolation and identification of OPCs. Themethods of this invention may be used to isolate PDGFRα⁺ cells fromPDGFRα⁻ cells using an PDGFRα antibody or other reagent thatspecifically binds to PDGFRα by combining a population of neural orneural-derived cells which contains a fraction of OPCs with the antibodyor reagent, and then selecting for PDGFRα⁺ cells, to produce a selectedpopulation enriched in PDGFRα⁺ OPCs as compared with the population ofneural or neural-derived cells before selection.

The population of cells from which OPCs are isolated is preferably aneural tissue, a population of cells dissociated from neural tissue, apopulation of cells that can give rise to neural cells or neural tissue,or a population of cells in cell culture, e.g., cells in a neurosphereculture or an adherent neural stem cell culture. Identification ofoligodendrocyte precursor cell (OPC) involves contacting a population ofcells or neural cells (or tissue which contains neural or neural-derivedcells) with a reagent that binds to cell surface markers expressed bythe target population of OPCs. For example, the method may comprisecontacting a population of neural or neural-derived cells with a reagentthat binds to PDGFRα (e.g., a monoclonal antibody) and detecting thecontact between the reagent that binds to PDGFRα and PDGFRα on thesurface of cells. Target OPCs are included in the population of cellsthat reagent binds to the reagent. The identity of those cells can beconfirmed by any assays known on the art to demonstrate that the cellsare, in fact, OPCs, i.e., capable of proliferation and capable ofdifferentiating into mature oligodendrocytes.

The OPCs according to some embodiments may be further characterized asbeing immunonegative for CD105 (CD105⁻). Accordingly, the method ofidentifying, isolating, or enriching populations of oligodendrocyteprecursor cells may additionally comprise selecting from a population ofneural or neural-derived cells for cells that are immunonegative forCD105 (CD105⁻ cells) and eliminating the CD105⁺ cells from thepopulation. Selection of CD105⁻ cells may then be followed by selectingfrom the remaining population for at least one cell that isimmunopositive for PDGFRα (PDGFRα⁺). In the alternative, the inventionprovides methods for isolating a oligodendrocyte precursor cell (OPC),by selecting from a population of neural or neural-derived cells forcells that are immunopositive for PDGFRα (PDGFRα⁺), e.g., binds tomonoclonal antibody PDGFRα; eliminating the non-immunoreactive (PDGFRα⁻)cells from the population; and selecting from the remaining populationfor cells that are immunonegative for CD105 (CD105⁻), i.e., eliminatingCD105⁺ cells from the population.

The OPCs according to some embodiments may be further characterized asbeing immunopositive for CD133 (CD133⁺). Accordingly, the method ofidentifying, isolating, or enriching populations of oligodendrocyteprecursor cells may additionally comprise selecting from a population ofneural or neural-derived cells for cells that are immunopositive forCD133 (CD133⁺ cells) and eliminating the CD133⁻ cells from thepopulation. Selection of CD133⁺ cells may then be followed by selectingfrom the remaining population for at least one cell that isimmunopositive for PDGFRα (PDGFRα⁺).

Accordingly, the invention further provides for the enrichment of targetOPCs from neural tissue or neural stem cell cultures (e.g., suspensioncultures or adherent cultures). The methods and composition of theinvention are thus useful for the enrichment of target OPC from neuraltissue in which stem cells and progenitor cells occur at low frequency,or may have been depleted, such as late embryo, juvenile, and adulttissue. Thus, one of skill in the art can combine a population of neuralor neural-derived cells containing a fraction of target OPCs with areagent that specifically binds to, for example, PDGFRα, and then selectfor the PDGFRα⁺ cells. In this way, the selected PDGFRα⁺ cells areenriched in the fraction of OPC as compared with the population ofneural or neural-derived cells. According to preferred embodiments, thetarget OPCs may be characterized based on a medium to high expression ofPDGFRα (e.g., PDGFRα^(med) or PDGFRα^(high)). For example, target OPCsare included in the PDGFRα⁺ cells sorted from suspended neurospheresbased on medium to high expression (e.g., PDGFRα^(med) orPDGFRα^(high)). Target OPCs may be further identified by theirexpression of the markers CD105, CD133, A2B5, PSA-NCAM, O4, and/or NG2in accordance with the present invention.

Any method for selecting a population of cells on the basis of cellmarker expression known in the art may be used to select for the OPCs ofthe present invention. For example, the identification of PDGFRα⁺ and/orCD133⁺ target cell populations may involve contacting a population ofneural cells (or tissue which contains neural or neural derived cells)with a reagent that binds to PDGFRα and/or CD133, and detecting thecontact between the reagent that binds to PDGFRα and/or CD133 and PDGFRαand/or CD133 on the surface of cells. Target OPCs are included in thepopulation of those cells to which the reagent binds. The identity ofthose cells can be confirmed by assays to demonstrate that the cellsare, in fact, OPCs, i.e., they are capable of differentiating intomature oligodendrocytes. Use of traditional techniques for cell sorting,such as by immunoselection (e.g., fluorescence activated cell separation(FACS)), permits identification, isolation, and/or enrichment for cellsin which contact between the reagent and the PDGFRα antigen has beendetected.

One of skill in the art can introduce an isolated target OPC(s) to aculture medium; proliferate the isolated target OPC(s) in culture;culture the progeny of the isolated target OPC(s) under conditions inwhich the isolated target OPC(s) differentiates into oligodendrocytes;and detect the presence of oligodendrocytes. The presence ofoligodendrocytes characterizes the isolated target OPC(s) as an OPC.

Any cell markers known in the art may also be used for the positive andnegative selection of OPCs. For example, monoclonal antibodies (mAb)against human CD45 may be used to exclude blood cell contamination infetal tissue. In some cases, mAb against human CD34 may be used toexclude endothelial cells and endothelial-neural progenitor complexes.In some cases, antibodies against human CD24 may be used to excludethose cells that are not likely to initiate neurospheres. Any of theseantibodies may be used alone, in combination, or sequentially in themethods for enriching the target cell populations disclosed herein.

Using the techniques and methods disclosed herein, one of skill in theart can derive the population of target OPCs by immunoselection usingthe appropriate antibody or series of preferred antibodies. According tosome embodiments, the population of target cells contains at least 30%PDGFRα⁺ OPCs, preferably at least 50-70% PDGFRα⁺ OPCs, and morepreferably greater than 90% PDGFRα⁺ OPCs (e.g., 92% or more, 94% ormore, 96% or more, or 98% or more). Most preferable would be asubstantially pure population of PDGFRα⁺ OPCs, comprising at least 95%PDGFRα⁺ OPCs (e.g., 97% or 99%).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, CD105⁻ OPCs, preferably at least 50-70% PDGFRα⁺,CD105⁻ OPCs, and more preferably greater than 90% PDGFRα⁺, CD105⁻ OPCs(e.g., 92% or more, 94% or more, 96% or more, or 98% or more). Mostpreferable would be a substantially pure population of PDGFRα⁺, CD105⁻OPCs, comprising at least 95% PDGFRα⁺, CD105⁻OPCs (e.g., 97% or 99%).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, A2B5⁻ OPCs, preferably at least 50-70% PDGFRα⁺,A2B5⁻ OPCs, and more preferably greater than 90% PDGFRα⁺, A2B5⁻ OPCs(e.g., 92% or more, 94% or more, 96% or more, or 98% or more). Mostpreferable would be a substantially pure population of PDGFRα⁺, A2B5⁻OPCs, comprising at least 95% PDGFRα⁺, A2B5⁻ OPCs (e.g., 97% or 99%).According to some embodiments, the population of target cells isadditionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, A2B5^(lo/−) OPCs, preferably at least 50-70%PDGFRα⁺, A2B5^(lo/−) OPCs, and more preferably greater than 90% PDGFRα⁺,A2B5^(lo/−) OPCs (e.g., 92% or more, 94% or more, 96% or more, or 98% ormore). Most preferable would be a substantially pure population ofPDGFRα⁺, A2B5^(lo/−) OPCs, comprising at least 95% PDGFRα⁺, A2B5^(lo/−)OPCs (e.g., 97% or 99%). According to some embodiments, the populationof target cells is additionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα^(med/high), A2B5⁻ OPCs, preferably at least 50-70%PDGFRα^(med/high), A2B5⁻ OPCs, and more preferably greater than 90%PDGFRα^(med/high), A2B5⁻ OPCs (e.g., 92% or more, 94% or more, 96% ormore, or 98% or more). Most preferable would be a substantially purepopulation of PDGFRα^(med/high), A2B5⁻ OPCs, comprising at least 95%PDGFRα^(med/high), A2B5⁻ OPCs (e.g., 97% or 99%). According to someembodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα^(med/high), A2B5⁻ OPCs, preferably at least 50-70%PDGFRα^(med/high), A2B5^(lo/−) OPCs, and more preferably greater than90% PDGFRα^(med/high), A2B5^(lo/−) OPCs (e.g., 92% or more, 94% or more,96% or more, or 98% or more). Most preferable would be a substantiallypure population of PDGFRα^(med/high), A2B5^(lo/−) OPCs, comprising atleast 95% PDGFRα^(med/high), A2B5⁻ OPCs (e.g., 97% or 99%). According tosome embodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, PSA-NCAM⁻ OPCs, preferably at least 50-70%PDGFRα⁺, PSA-NCAM⁻ OPCs, and more preferably greater than 90% PDGFRα⁺,PSA-NCAM⁻ OPCs (e.g., 92% or more, 94% or more, 96% or more, or 98% ormore). Most preferable would be a substantially pure population ofPDGFRα⁺, PSA-NCAM⁻ OPCs, comprising at least 95% PDGFRα⁺, PSA-NCAM⁻ OPCs(e.g., 97% or 99%). According to some embodiments, the population oftarget cells is additionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, PSA-NCAM^(lo/−) OPCs, preferably at least 50-70%PDGFRα⁺, PSA-NCAM^(lo/−) OPCs, and more preferably greater than 90%PDGFRα⁺, PSA-NCAM^(lo/−) OPCs (e.g., 92% or more, 94% or more, 96% ormore, or 98% or more). Most preferable would be a substantially purepopulation of PDGFRα⁺, PSA-NCAM^(lo/−) OPCs, comprising at least 95%PDGFRα⁺, PSA-NCAM^(lo/−) OPCs (e.g., 97% or 99%). According to someembodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα^(med/high), PSA-NCAM⁻ OPCs, preferably at least50-70% PDGFRα^(med/high), PSA-NCAM⁻ OPCs, and more preferably greaterthan 90% PDGFRα^(med/high), PSA-NCAM⁻ OPCs (e.g., 92% or more, 94% ormore, 96% or more, or 98% or more). Most preferable would be asubstantially pure population of PDGFRα^(med/high), PSA-NCAM⁻ OPCs,comprising at least 95% PDGFRα^(med/high), PSA-NCAM⁻ OPCs (e.g., 97% or99%). According to some embodiments, the population of target cells isadditionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα^(med/high), PSA-NCAM^(lo/−) OPCs, preferably atleast 50-70% PDGFRα^(med/high), PSA-NCAM^(lo/−) OPCs, and morepreferably greater than 90% PDGFRα^(med/high), PSA-NCAM^(lo/−) OPCs(e.g., 92% or more, 94% or more, 96% or more, or 98% or more). Mostpreferable would be a substantially pure population ofPDGFRα^(med/high), PSA-NCAM^(lo/−) OPCs, comprising at least 95%PDGFRα^(med/high), PSA-NCAM^(lo/−) OPCs (e.g., 97% or 99%). According tosome embodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, CD133⁺, A2B5^(lo/−) OPCs, preferably at least50-70% PDGFRα⁺, CD133⁺, A2B5^(lo/−) OPCs, and more preferably greaterthan 90% PDGFRα⁺, CD133⁺, A2B5^(lo/−) OPCs (e.g., 92% or more, 94% ormore, 96% or more, or 98% or more). Most preferable would be asubstantially pure population of PDGFRα⁺, CD133⁺, A2B5^(lo/−) OPCs,comprising at least 95% PDGFRα⁺, CD133⁺, A2B5^(lo/−) OPCs (e.g., 97%,99%). According to some embodiments, the population of target cells isadditionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, CD133⁺, PSA-NCAM^(lo/−) OPCs, preferably at least50-70% PDGFRα⁺, CD133⁺, PSA-NCAM^(lo/−) OPCs, and more preferablygreater than 90% PDGFRα⁺, CD133⁺, PSA-NCAM^(lo/−) OPCs (e.g., 92% ormore, 94% or more, 96% or more, or 98% or more). Most preferable wouldbe a substantially pure population of PDGFRα⁺, CD133⁺, PSA-NCAM^(lo/−)OPCs, comprising at least 95% PDGFRα⁺, CD133⁺, PSA-NCAM^(lo/−) OPCs(e.g., 97%, 99%). According to some embodiments, the population oftarget cells is additionally immunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs, preferably atleast 50-70% PDGFRα⁺, A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs, and morepreferably greater than 90% PDGFRα⁺, A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs(e.g., 92% or more, 94% or more, 96% or more, or 98% or more). Mostpreferable would be a substantially pure population of PDGFRα⁺,A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs, comprising at least 95% PDGFRα⁺,A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs (e.g., 97%, 99%). According to someembodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα^(med/high), A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs,preferably at least 50-70% PDGFRα^(med/high), A2B5^(lo/−),PSA-NCAM^(lo/−) OPCs, and more preferably greater than 90%PDGFRα^(med/high), A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs (e.g., 92% or more,94% or more, 96% or more, or 98% or more). Most preferable would be asubstantially pure population of PDGFRα^(med/high), A2B5^(lo/−),PSA-NCAM^(lo/−) OPCs, comprising at least 95% PDGFRα^(med/high),A2B5^(lo/−), PSA-NCAM^(lo/−) OPCs (e.g., 97%, 99%). According to someembodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻).

According to some embodiments, the population of target cells containsat least 30% PDGFRα⁺, O4⁻ OPCs, preferably at least 50-70% PDGFRα⁺, O4⁻OPCs, and more preferably greater than 90% PDGFRα⁺, O4⁻ OPCs (e.g., 92%or more, 94% or more, 96% or more, or 98% or more). Most preferablewould be a substantially pure population of PDGFRα⁺, O4⁻ OPCs,comprising at least 95% PDGFRα⁺, O4⁻ OPCs (e.g., 97%, 99%). According tosome embodiments, the population of target cells is additionallyimmunonegative for CD105 (CD105⁻). The degree of enrichment obtained,and actually used, depends on a number of factors, including the methodof selection, the method of growth, and/or the dose of the cells thatare placed in culture.

Cryopreservation and Handling

According to some embodiments, the OPCs of the present embodiments maybe cryopreserved according to routine procedures. In some embodiments,cryopreserving involves freezing about one to ten million cells in“freeze” medium, which may comprise proliferation medium andantioxidants such as NAC (0.1 to 2 mM; e.g., 0.5 mM, 1 mM, etc.).Proliferation medium is preferably absent the growth factor mitogens.For example, suspended cells may be centrifuged and any growth medium isaspirated and replaced with freeze medium. Cells may then be slowlyfrozen, by, e.g., placing in a container at −80° C. or frozen in liquidnitrogen. Cells are thawed by swirling in a 37° C. bath, resuspended infresh proliferation medium, and grown as usual.

According to some embodiments, the OPCs of the present embodiments maybe cryopreserved in a ready to use format (such as a pharmaceuticalgrade vial or container). In some embodiments, the OPCs are thawed andcultured prior to use. In some embodiments, the OPCs are thawed andcultured in suspension prior to use. In some embodiments, the OPCs arethawed and cultured on an adherent substrate prior to use. The periodfor culturing after thawing may be 1 to 24 hours. In some embodiments,the period for culture after thawing may be from 1 to 2 days.

According to some embodiments, the OPCs of the present invention may beheld in a suspension culture format. According to some embodiments, theOPCs of the present invention may be held in a suspension culture formatafter detachment from a culture plate (e.g., post trypsin treatment).For example, adherent OPCs are detached by treatment with trypsin andare transferred to a suspension culture medium. The period of time inwhich the OPCs are held in suspension may be referred to as the “hold”period. The hold period in suspension is advantageous for at least thefollowing 3 reasons: 1) would allow cells to recover from a potentiallydamaging enzyme treatment prior to transplantation and/orcryopreservation; 2) it would introduce more flexibility in animalsurgery scheduling and 3) would make shipment of ready-to-transplantOPCs to an off-site location (laboratory or clinic) possible. Accordingto some embodiments, the hold period may be 2, 4, 6, 8, 12, 18, or 24hours. According to some embodiments, the hold period may be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 days.

Tissue Source

Any suitable tissue source may be used to derive the OPCs of thisinvention. The adult human CNS has been shown to contain oligodendrocyteprecursor cells that are capable of proliferating, and which couldmature into myelinating oligodendrocytes under the appropriateconditions. Accordingly, the population of cells can be derived fromlate embryo, juvenile, or adult mammalian CNS tissue, or it may bederived from existing cultures of neural stem cells, as described inWeiss, U.S. Pat. No. 5,750,376, or Johe, U.S. Pat. No. 5,753,506. TheOPCs may also be obtained from any tissue or cellular source that iscapable of giving rise to neural tissue. In one preferred embodiment,the OPCs are human.

OPCs may be been isolated from neural or neural-derived cells of severalmammalian species including, but not limited to, mice, rats, pigs,non-human primates, and humans. Neural or neural-derived cells may beobtained from embryonic, fetal, post-natal, juvenile, or adult neuraltissue, which includes brain and spinal cord. For example, neural orneural-derived cells may be obtained from the cerebral cortex,cerebellum, midbrain, brainstem, spinal cord, and ventricular tissue, aswell as areas of the PNS including the carotid body and the adrenalmedulla. Other preferred areas include regions in the basal ganglia,preferably the striatum, which consists of the caudate and putamen, orvarious cell groups such as the globus pallidus, the subthalamicnucleus, the nucleus basalis, the substantia nigra pars compacta, aswell as from ventricular tissue found lining CNS ventricles, includingthe subependyma. The subventricular zone and ventral neuroepithelium arepreferred source of OPCs in the adult animal.

In addition to OPCs, a population of cells exists within the adult CNSthat exhibit stem cell properties in their ability to self-renew and toproduce the differentiated mature cell phenotypes of the adult CNS suchas oligodendrocytes. These stem cells are found throughout the CNS,particularly in the subventricular region and the dentate gyrus of thehippocampus, and represent a source of neural or neural derived cellsfrom which the target OPCs may be isolated. Neural stem cells have alsobeen isolated from a variety of adult CNS ventricular regions, includingthe frontal lobe, conus medullaris, thoracic spinal cord, brain stem,and hypothalamus.

Growth factor-responsive stem cells can be isolated from many regions ofthe neuraxis and at different stages of development, of murine, rodent,mammalian, and human CNS tissue. These cells vary in their response togrowth factors such as EGF, basic FGF (bFGF, FGF-2) and transforminggrowth factor alpha (TGFα) and can be maintained and expanded in culturein an undifferentiated state for long periods of time. (See, e.g.WO93/01275 and WO94/16788, incorporated herein by reference).

Proliferation

OPCs can be induced to proliferate either by culturing the cells insuspension or on an adherent substrate. See, e.g., U.S. Pat. Nos.5,750,376 and 5,753,506 (both incorporated herein by reference in theirentirety), and medium described therein. Both allografts and autograftsare contemplated for transplantation purposes.

Typically, OPCs of the present embodiments are cultured in a medium thatpermits their growth and proliferation. The culture in which theisolated OPCs proliferates can be a serum-free medium containing one ormore predetermined growth factors effective for inducing proliferation.The culture medium may be supplemented with a growth factor selectedfrom platelet-derived growth factor (PDGF), epidermal growth factor(EGF), basic fibroblast growth factor (FGF-2; bFGF), NT3, IGF1 orcombinations thereof. The culture medium may be further supplementedwith N2 and B27. The conditions in which the OPCs differentiate tooligodendrocytes include culturing the OPC progeny on a laminin orlaminin plus fibronectin-coated surface in culture medium containingfetal bovine serum (FBS) or T3 (triiodothyronine) without EGF, bFGF,PDGF, NT3, IGF1 or LIF.

According to some embodiments, the OPCs of the present embodiments maybe passaged from 1 to 20 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and 20 times) post isolation from theprimary tissue source and may be induced to proliferate by culturing thecells in suspension or on an adherent substrate. Passaging (a.k.a,subculturing or splitting) typically involves detaching cells from thesurface of the primary culture vessel by trypsinization or mechanicalmeans. The resultant cell suspension is then subdivided, or reseeded,into fresh cultures. Secondary cultures are checked for growth and fedperiodically, and may be subsequently subcultured to produce tertiarycultures and so on. The time between passaging of cells varies anddepends on the growth rate.

The proliferation medium can be any medium known in the art to induceproliferation of the OPCs without inducing their differentiation.Example 3 provided herein provides an exemplary medium for proliferatingthe OPCs of the present embodiments. Cell passage or splitting isnecessary to maintain cells in exponential growth. Methods for passagingor splitting cells are well known in the art. The OPCs may be passagedusing any known method known in the art.

Differentiation

When OPCs are cultured under conditions that allow differentiation,progenitor cells differentiate to oligodendrocytes. Differentiation ofthe cells can be induced by any method known in the art, which includethe liberation of inositol triphosphate and intracellular Ca²⁺,liberation of diacyl glycerol, and the activation of protein kinase Cand other cellular kinases, and the like. Treatment with phorbol esters,differentiation-inducing growth factors, hormones and other chemicalsignals can induce differentiation. Differentiation can be induced bygrowth factor exhaustion, for example, by removal of mitogens, byleaving the cells in culture without media renewal, or by absence ofpassaging.

The induction of proliferation (and differentiation) of the OPCs can bedone either by culturing the cells in suspension or on a substrate ontowhich they can adhere. Alternatively, proliferation and differentiationof OPCs can be induced, under appropriate conditions, in the host in thefollowing combinations: (1) proliferation and differentiation in vitro,then transplantation, (2) proliferation in vitro, transplantation, thenfurther proliferation and differentiation in vivo, (3) proliferation invitro, transplantation and differentiation in vivo, and (4)proliferation and differentiation in vivo. Proliferation anddifferentiation in vivo or in situ can involve a non-surgical approachthat coaxes OPCs to proliferate in vivo with pharmaceuticalmanipulation. Such methods involving the transplantation of OPCs arediscussed in further detail below.

Use of Purified Stem Cell/Progenitor Cells.

The target OPC populations identified using the methods described hereinare useful in a variety of ways, including for drug screening,diagnostics, transplantation, and treatment. The OPCs may be used toreconstitute a host whose cells have been lost through disease orinjury. Genetic diseases associated with cells may be treated by geneticmodification of autologous or allogeneic OPCs to correct a geneticdefect or treat to protect against disease. Alternatively, normalallogeneic OPCs may be transplanted. Diseases other than thoseassociated with cells may also be treated, where the disease is relatedto the lack of a particular secreted product such as hormone, enzyme,growth factor, or the like.

CNS disorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, ischemia, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include, for example, Alzheimer'sDisease, Multiple Sclerosis (MS), Huntington's Disease, AmyotrophicLateral Sclerosis, and Parkinson's Disease, have been linked to thedegeneration of neural cells in particular locations of the CNS, leadingto the inability of these cells or the brain region to carry out theirintended function. By providing for maturation, proliferation anddifferentiation into oligodendrocytes through specific different growthfactors, the oligodendrocyte progenitor cells may be used as a source ofoligodendrocytes.

The target OPC populations may also be used in the isolation andevaluation of factors associated with the differentiation and maturationof cells. Thus, the cells may be used in assays to determine theactivity of media, such as conditioned media, evaluate fluids for growthfactor activity, involvement with dedication of lineages, or the like.

The target OPC populations may be frozen at liquid nitrogen temperaturesand stored for long periods of time, being thawed and capable of beingreused. The cells will usually be stored in 7.5% DMSO and 4% HSA (humanserum albumin). Once thawed, the cells may be expanded by use of growthfactors or cells associated with OPC proliferation and differentiation.

Transplantation

The target OPC populations obtained from neural cell populations orneural tissue may be introduced (e.g., by transplantation) into amammal, particularly to compensate for lost or dysfunctionaloligodendrocytes. The mammal is preferably a human, canine, feline,rodent, sheep, goat, cattle, horse, pig, or non-human primate. Mostpreferably, the mammal is human. Since OPCs may be cultured from braintissues from mammals of any age, including adults, it is preferable togrow neural stem cells using a mammal's own tissue for autologoustransplantation. Allogeneic and xenogeneic transplantations are alsopossible, particularly when the transplantation site is in the brain oreye, where immunologic rejection is less severe due to the blood-brainor blood-retina barrier.

In some embodiments, the OPCs of the present embodiments aretransplanted at a dose of at least on the order of greater than 1×10²⁰total nucleated cells, or at least on the order of 10¹⁹, or 10¹⁸, or10¹⁷, or 10¹⁶, or 10¹⁵, or 10¹⁴, or 10¹³, or 10¹², or 10¹¹, or 10¹⁰, or10⁹, or 10 ⁸, or 10⁷, or 10⁶, or 10⁵ cells. In some embodiments, theOPCs of the present embodiments may be transplanted at a dose of between1×10⁶ to 1×10¹², 1×10⁶ to 1×10⁹, 1×10⁸ to 1×10¹⁰, 1×10⁹ to 1×10¹², and1×10⁹ to 1×10¹⁰ cells. In some embodiments, the dosage of cells isprepared in a sealed, pharmaceutical quality vial in a format that isready to administer to a subject.

It is also that the target OPCs may be transplanted into a mammal andinduced to form oligodendrocytes in vivo. Thus, target OPC populationsmay be expanded in culture using established methods, transplanted intothe mammal, and contacted in vivo with the oligodendrocyte promotingfactor to produce oligodendrocytes. Optionally, the transplanted OPCscan be expanded again in vivo by administering to the mammal anybiological agents known to increase the number of OPCs.

According to some embodiments, the OPCs of the present invention aretransplanted in between 1 to 5 days post passaging, preferably between 1to 2 days past passaging. The OPCs of the present embodiments may betransplanted as clusters or disassociated cell suspensions. Engraftmentdata (not shown) using cells obtained in this manner are healthy andresult in graft containing high number of myelinating oligodendrocytes.According to some embodiments, the OPCs of the present embodiments maybe held in suspension between 10 minutes to 5 days (e.g., 30 minutes, 1hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours,3 days, 4 days etc.) post passaging in a pharmaceutical quality vial ina format that is ready to administer to a subject. In some the OPCs ofthe present embodiments may be held in suspension at a dose of at leaston the order of greater than 1×10²⁰ total nucleated cells, or at leaston the order of 10¹⁹, or 10 ¹⁸, or 10¹⁷, or 10¹⁶, or 10¹⁵, or 10¹⁴, or10¹³, or 10¹², or 10¹¹, or 10¹⁰, or 10⁹, or 10⁸, or 10 ⁷, or 10⁶, or 10⁵cells. In some embodiments, the OPCs of the present embodiments may beheld in suspension at a dose of between 1×10⁶ to 1×10¹², 1×10⁶ to 1×10⁹,1×10⁸ to 1×10¹⁰, 1×10⁹ to 1×10¹², and 1×10⁹ to 1×10¹⁰ cells. In someembodiments, the dosage of cells is prepared in a sealed, pharmaceuticalquality vial in a format that is ready to administer to a subject.

The oligodendrocyte promoting factors or the biological agents may beadministered by any suitable route established in the art, including,for example, orally, topically, rectally, vaginally, intrathecally,intravascularly, intravenously, intramuscularly, intraperitoneally,transdermally, intradermally, subcutaneously, nasally or by inhalation.The route of administration depends primarily on the nature of theagent. For example, GM-CSF is capable of crossing the blood-brainbarrier, hence it can be administered systemically as well as into thebrain. The preferred method of administration is injection (e.g., with aneedle or a catheter) or infusion.

The target OPCs may be transplanted “naked” into patients according toconventional techniques, into the CNS as described, for example, in U.S.Pat. Nos. 5,082,670 and 5,618,531, the disclosures of which areincorporated herein by reference, or into any other suitable site in thebody. In some embodiments, the OPCs are transplanted directly into theCNS. Parenchymal and intrathecal sites are contemplated. It will beappreciated that the exact location in the CNS will vary according tothe disease state.

According to some embodiments, the OPCs may be allowed to aggregateprior to implantation, or may be applied directly as dissociated singlecells. When using OPC aggregates, transplantation is preferablyperformed using small sized aggregates approximately 10-500 μm indiameter, preferably 40-50 μm in diameter. Preferably, from about 1million cells to about 1 billion cells are transplanted. For example, atotal of about 1 million, about 5 million, about 10 million, about 25million, about 50 million, about 75 million, about 100 million, about250 million, about 500 million, about 750 million, or about 1 billioncells are transplanted.

The OPCs are preferably introduced into the brain or spinal cord of themammal, particularly at sites where oligodendrocytes are insufficientand/or dysfunctional, for example, around axons that have beendemyelinated. In humans, areas of demyelination are generally associatedwith plaque like structures, which can be visualized with magneticresonance imaging (MRI). The cells may also be transplanted into otherareas of the central nervous system. One particularly useful approach isto transplant into the “minor image” location of a target lesion in theother hemisphere, since cells are known to efficiently migrate to thecorresponding location in the opposite hemisphere through the corpuscallosum.

According to some embodiments, the OPCs are introduced directly toregions of the brain or spinal cord. Directed introduction of the OPCsmay be carried out using any methods known in the art. Thus, accordingto some embodiments, the OPCs are introducted to the target brain regionvia injection. Preferably, the OPCs are introduced into brain regionsthat are heavily myelinated (rich in white matter). The fimbria is aprominent band of white matter along the medial edge of the hippocampus.White matter forms the bulk of the deep parts of the brain and thesuperficial parts of the spinal cord. The corpus callosum is the largestwhite matter structure in the brain that connects the left and rightcerebral hemispheres. In some embodiments, the target brain regionsinclude the fimbria, callosum, cerebral peduncle, internal capsule,spinal cord, brain stem, motor cortex, olfactory cortex, somatosensorycortex, anterior cingulate gyrus, the Inferior temporal lobe, and theDorsolateral prefrontal cortex, and medulla oblongata.

Aggregates of gray matter such as the basal ganglia (caudate nucleus,putamen, globus pallidus, subthalamic nucleus, nucleus accumbens) andbrain stem nuclei (red nucleus, substantia nigra, cranial nerve nuclei)are spread within the cerebral white matter. Such areas are also targetbrain regions. Target brain regions include, but are not limited to, thetelencephalon (cerebral hemispheres, forebrain), diencephalon (thalamus,hypothalamus, epithalamus, prethalamus or subthalamus and pretectum),mesencephalon (midbrain), cerebellum, pons, and medulla oblongata. Themesencephalon includes the tectum (inferior colliculi and superiorcolliculi) and cerebral peduncle (midbrain tegmentum, crus cerebri,substantia nigra). The substantia nigra is part of the basal ganglia;the other parts of the basal ganglia include the striatum (caudatenucleus, putamen, and nucleus accumbens), globus pallidus, andsubthalamic nucleus. Target brain regions may include the brain stem,striatum, internal capsule, caudate nucleus and putamen.

Genetic Modification of OPCs.

The OPCs of the present embodiments may be genetically modified toprovide a therapeutically effective biologically active molecule. Insome embodiments, the genetically modified OPCs may be transplanted orintroduced to a subject in need thereof as described above.

In some embodiments, the OPCs of the present embodiments may begenetically modified to express a particular form of Myelin ProteolipidProtein (PLP), such as in the case of autologous transplant. Inaddition, the OPCs of the present embodiments may be geneticallymodified to express one or more of the following: telomerase (to preventtelomere erosion), growth factors, morphogens, enzymes, anti-apoptoticgenes (e.g., sonic hedgehog, FGF2, NT3, BDNF, PDGF, IGF, NGF),arylsuphatase A (metachromatic leukodystrophy), galactosylceramidase(krabbe's), superoxide dismutase and other proteins involved inantioxidant defense, and Bcl-XL.

The OPCs described herein can be genetically engineered or modifiedaccording to known methodology. The term “genetic modification” refersto the stable or transient alteration of the genotype of a cell byintentional introduction of exogenous DNA. DNA may be synthetic, ornaturally derived, and may contain genes, portions of genes, or otheruseful DNA sequences. The term “genetic modification” is not meant toinclude naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike.

A gene of interest (i.e., a gene that encodes a biologically activemolecule) can be inserted into a cloning site of a suitable expressionvector by using standard techniques. These techniques are well known tothose skilled in the art. See, e.g., WO 94/16718, incorporated herein byreference.

The expression vector containing the gene of interest may then be usedto transfect the desired cell line. Standard transfection techniquessuch as calcium phosphate co-precipitation, DEAE-dextran transfection,electroporation, biolistics, or viral transfection may be utilized.Commercially available mammalian transfection kits may be purchased frome.g., Stratagene. Human adenoviral transfection may be accomplished asdescribed in Berg et al. Exp. Cell Res., 192, pp. (1991). Similarly,lipofectamine-based transfection may be accomplished as described inCattaneo, Mol. Brain. Res., 42, pp. 161-66 (1996).

A wide variety of host/expression vector combinations may be used toexpress a gene encoding a biologically active molecule of interest. See,e.g., U.S. Pat. No. 5,545,723, herein incorporated by reference, forsuitable cell-based production expression vectors.

Increased expression of the biologically active molecule can be achievedby increasing or amplifying the transgene copy number usingamplification methods well known in the art. Such amplification methodsinclude, e.g., DHFR amplification (see, e.g., Kaufman et al., U.S. Pat.No. 4,470,461) or glutamine synthetase (“GS”) amplification (see, e.g.,U.S. Pat. No. 5,122,464, and European published application EP 338,841),all herein incorporated by reference.

Any expression vector known in the art may be used to express thebiologically active molecule. In some embodiments, alentivirally-derived vector may be particularly useful for the deliveryof exogenous genes. Such lentiviral vectors are known in the art. Insome embodiments, exogenous genes may need to be introduced into thetarget OPCs expression. Such genes may be under the control of aconstitutive or inducible promoters to effect optimal co-expression.Exogenous DNA may be introduced to a precursor cell by viral vectors(retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, lentivirus and the like) or direct DNAtransfection (lipofection, CaPO₄ transfection, DEAE-dextran,electroporation, and the like).

The OPCs of the present embodiments may be genetically modified for drugscreening purposes or for the purposes of detecting cells of theoligodendrocyte or OPC lineage. In some embodiments, OPCs may begenetically modified with one or more reporter genes. Such reportergenes include fluorescent proteins (e.g., green fluorescent proteins,yellow fluorescent proteins, blue fluorescent proteins, cyan fluorescentproteins, etc.), DsRed2, mCherry, tdTomato, and AmCyan1. Preferredpromoters include one or more of the following promoters: MBP, CNPase,Olig2, Sox10, Plp, and PDGFR promoter.

Treatment

A number of neurologic diseases are associated with defects inmyelination and in neuronal homeostasis and function. Examples of thesedemyelinating diseases or conditions or dysmyelinating disordersinclude, but are not limited to, multiple sclerosis (including therelapsing and chronic progressive forms of multiple sclerosis, acutemultiple sclerosis, neuromyelitis optica (Devic's disease)), diffusecerebral sclerosis (including Shilder's encephalitis periaxialis diffusaand Balo's concentric sclerosis). Demyelinating diseases also include avariety of diseases wherein demyelination is caused by viral infections,vaccines, spinal cord injury, and genetic disorders. Examples of thesedemyelinating diseases or dysmyelinating disorders include, but are notlimited to, acute disseminated encephalomyelitis (occurring aftermeasles, chickenpox, rubella, influenza or mumps; or after rabies orsmallpox vaccination), necrotizing hemorrhagic encephalitis (includinghemorrhagic leukoencephalitis), and leukodystrophies (including Krabbe'sglobboid leukodystrophy, metachromatic leukodystrophy,adrenoleukodystrophy, adrenomyeloneuropathy, adrenomyeloneuropathy,radiation induced myelination disorders, transverse myelitits,Pelizaeus-Merzbacher disease, Canavan's disease and Alexander'sdisease). The demyelinating disease or dysmyelinating disorders ispreferably multiple sclerosis, cerebral palsy, diffuse cerebralsclerosis, or Pelizaeus-Merzbacher disease (PMD), and, most preferably,Pelizaeus-Merzbacher disease.

The cells and methods of this invention may be useful in the treatmentof various neurodegenerative diseases, demyelinating diseases and/ordysmyelinating disorders. It is contemplated that the cells will replacediseased, damaged or lost tissue in the host. Alternatively, thetransplanted tissue may augment the function of the endogenous affectedhost tissue.

According to some embodiments, there is provided a method of enhancingoligodendrocyte production in vivo by administering the target OPCs to amammal under conditions that result in oligodendrocyte formation. Theresultant oligodendrocytes are capable of myelinating (or remyelinating)demyelinated neurons in the mammal, whereby dysmyelinating disordersand/or demyelinating diseases in the mammal can be treated orameliorated.

According to some embodiments, there is provided a method of enhancingoligodendrocyte production in vivo by identifying and isolating targetpopulations of OPCs, culturing the target populations of OPCs underconditions to promote their proliferations, and administering the OPCsto a mammal under conditions that result in oligodendrocyte formation.The resultant oligodendrocytes are capable of myelinating (orremyelinating) demyelinated neurons in the mammal, wherebydysmyelinating disorders and/or demyelinating diseases in the mammal canbe treated or ameliorated.

According to some embodiments, there is provided a method of enhancingoligodendrocyte production in vivo by identifying and isolating targetpopulations of OPCs, culturing the target populations of OPCs underconditions to promote their proliferations, differentiating the targetpopulations of OPCs into oligodendrocytes, and administering theoligodendrocytes to a mammal under conditions that result inoligodendrocyte engraftment. The resultant oligodendrocytes are capableof myelinating (or remyelinating) demyelinated neurons in the mammal,whereby dysmyelinating disorders and/or demyelinating diseases in themammal can be treated or ameliorated.

According to some embodiments, there is provided a method of enhancingoligodendrocyte production in vivo by identifying and isolating targetpopulations of OPCs and administering target OPCs to a mammal underconditions that result in oligodendrocyte engraftment. The resultantoligodendrocytes are capable of myelinating (or remyelinating)demyelinated neurons in the mammal, whereby dysmyelinating disordersand/or demyelinating diseases in the mammal can be treated orameliorated.

Drug Screening

The OPCs of the present invention may also be used in a method of drugscreening or drug discovery. Any cell-based drug screening protocolknown in the art may be used in conjunction with the OPCs of the presentinvention. A wide variety of assays may be used for this purpose,including toxicology testing; immunoassays for protein binding;determination of cell growth, differentiation and functional activity;production of hormones; and the like. The assays may be performed invitro, in situ, in vivo, and ex vivo. For example, the OPCs of thepresent invention may be used in a drug screening method comprising thesteps of a) selecting from an enriched target OPC population, b)engrafting a non-human mammal with the resulting enriched population; c)administering a test compound to the non-human mammal; and d) comparingthe effect of administration of said test compound in the engraftedmammal with a control non-human mammal not administered said testcompound.

According to some embodiments, the present invention provides a methodof screening for compounds that affect a biological function of anenriched population of target oligodendrocyte precursor cellscomprising: (a) contacting an enriched population of targetoligodendrocyte precursor cells obtained by the method of claim 1 with atest compound; and (b) detecting a change in a biological function ofthe oligodendrocyte precursor cells. The change in biological functionmay include, but is not limited to, changes in one or more of thefollowing: myelination, differentiation into oligodendrocytes,proliferation rate, cell migration, cell viability, gene expression,protein expression, protein levels in the culturing medium,dedifferentiation, growth characteristics, and/or cell morphology.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples. The change inparameters in response to the agent is measured, and the resultevaluated by comparison to reference cultures, e.g. in the presence andabsence of the agent, obtained with other agents, etc. The agents may beconveniently added in solution, or readily soluble form, to the mediumof cells in culture. The agents may be added in a flow-through system,as a stream, intermittent or continuous, or alternatively, by adding abolus of the compound, singly or incrementally, to an otherwise staticsolution. In a flow-through system, two fluids are used, where one is aphysiologically neutral solution, and the other is the same solutionwith the test compound added. The first fluid is passed over the cells,followed by the second. In a single solution method, a bolus of the testcompound is added to the volume of medium surrounding the cells. Theoverall concentrations of the components of the culture medium shouldnot change significantly with the addition of the bolus, or between thetwo solutions in a flow through method.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label a molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive,enzymatically active, etc., particularly a molecule specific for bindingto the parameter with high affinity fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells. Thus, antibodies can bemodified to provide a fluorescent dye as part of their structure.Depending upon the label chosen, parameters may be measured using otherthan fluorescent labels, using such immunoassay techniques asradioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.The quantization of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques.

Encapsulation

Any encapsulation protocol known in the art may be used with the OPCs ofthe present invention. The OPCs of the present invention may beencapsulated and used to deliver biologically active molecules,according to known encapsulation technologies, includingmicroencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and5,084,350, incorporated herein by reference), macroencapsulation (see,e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733 andpublished PCT patent applications WO 92/19195, WO 95/05452, eachincorporated herein by reference).

If the OPCs are encapsulated, macroencapsulation as described in U.S.Pat. Nos. 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 andpublished PCT patent application WO 95/05452, each incorporated hereinby reference is preferred. Cell number in the devices can be varied.Preferably each device contains between 10³-10⁹ cells, most preferably10⁵-10⁷ cells. A large number of macroencapsulation devices may beimplanted in the patient; preferably between one to 10 devices.

The following examples are illustrative, but not limiting, of themethods and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in therapy and that are obvious to thoseskilled in the art are within the spirit and scope of the embodiments.

Example 1 Selection of PDGFR Positive Cells

Fetal brain (16-20 weeks of gestation) was enzymatically treated with acombination of collagenase/hyaluronidase and trypsin to generate asingle cell suspension. Cells were ressuspended in Hank's balanced saltsolution containing 1 mM Sodium Pyruvate and 0.1% human serum albumin(staining buffer) and stained with the CD133 antibody. CD133⁺ cells wereaseptically sorted using a BD Vantage flow cytometer, under enrichmentmode. CD133⁺ enriched fraction was centrifuged, ressuspended in stainingbuffer and incubated with a 1:100 dilution of rabbit anti PDGFRαpolyclonal antibody (IgG) for 2 hours at 4° C. After two rinses instaining buffer, PDGFRα labeled cells were incubated for polyclonal goatanti-rabbit IgG-FITC antibody (Caltag). PDGFRα positive (FITC labeled)cells were aseptically sorted using a BD Vantage flow cytometer, underpurity mode. Sorted cells were cultured on poly-L-ornithine, laminin andfibronectin coated culture flasks in DMEM medium supplemented with B27,N2, NAC, L-Glutamine, Na Pyruvate, FGF2, PDGF-AA and NT3 (completemedium), with or without IGF1. Cell passaging was achieved by mildtrypsin treatment and re-plating in the same medium.

An alternative method used for the purification of PDGFRα positive cellsis to stain total brain cells (with or without enrichment for CD133)with the mouse monoclonal antibody 1:50 PDGFRα-PE (Pharmingen) for 2 hat 4° C., followed by purity aseptic sorting using a BD Aria flowcytometer.

Example 2 Selection of CD105 Negative Cells

The use of CD105⁻ cells as a selection marker is based on the presentinventors discovery that some cultures of purified PDGFRα⁺ cells expandat a much faster rate then others. Such accelerated growth was generallyaccompanied by the appearance of a cell type with a morphology distinctfrom that of oligodendrocyte progenitors. Based on the morphology andaccelerated growth in culture, it is likely that these cells are PDGFRα⁺fibroblasts; the growth advantage of fibroblasts in FGF2 containingmedia is well documented. Moreover, apart from exhausting mitogens,fibroblasts may also negatively condition the culture medium, affectingthe growth kinetics and the differentiation process of oligodendrocyteprogenitors into oligodendrocytes, both in vitro and in vivo. Thepresence of these fibroblastic cells therefore reduces the efficiency inobtaining an expanded culture of oligodendrocyte progenitors.

It is desirable to define a more pure and homogeneous oligodendrocyteprogenitor population, especially in the case when contaminating cellshave growth advantage. Therefore a search was initiated for cell surfacemarkers expressed specifically in fibroblasts that could be used todistinguish them from PDGFRα⁺ oligodendrocyte progenitors. A panel ofantibodies was used to stain cultures containing a mixture offibroblasts and oligodendrocytes. The results showed that the monoclonalantibody to CD105, which recognizes the glycoprotein endoglin, is auseful reagent to subdivide the PDGFRα⁺ population into twosubpopulations: PDGFRα⁺ CD105⁺ (fibroblasts) and PDGFRα⁺ CD105⁻(oligodendrocyte progenitors).

The sorting protocol for the isolation of fetal derived humanoligodendrocyte progenitors includes two antibodies, CD105-APC andPDGFRα-PE. Various cell lots have been generated using this two antibodyprotocol. The results indicate that these cell lots have similar growthcharacteristics and oligodendrocyte differentiation potential. Further,the appearance of fibroblasts is not observed in these cultures, up topassage 15, the highest passage tested. The results demonstrate thatCD105 is a useful negative selection marker for use in obtaining adesirable population of oligodendrocyte progenitors.

Example 3 Media for Proliferating OPCs

Proliferation medium was prepared with the following components in theindicated concentrations: Component Final Concentration DMEM, glutamine(Invitrogen, cat#25030-081) 2 mM, Na Pyruvate (Sigma, cat#S8636), 1 mM,NAC (Sigma, cat#A9165), 1 mM, N2 supplement (Invitrogen, cat#17502-048;containing transferrin, insulin, putrescine, selenium and progesterone),B27 supplement (Invitrogen, cat #17504-044), 20 ng/ml human bFGF(Biosource, cat#PHG0024), 20 ng/ml PDGF-AA (Peprotech, cat#100-13A) 10ng/ml NT3 (Peprotech, cat#450-03), 100 ng/ml IGF1 (Peprotech, cat#AF-100-11).

Example 4 Differentiation of OPCs

In a first differentiation protocol, proliferating OPCs are induced todifferentiate by physical removal or exhaustion of the growth factormitogens from the cell culture with addition of triiodothyronine (T3).

The staining protocol for oligodendrocytes was as follows:

O4 Staining for Oligodendrocytes. Cells are incubated with primaryantibodies to O4 (hybridoma supernatant, mouse monoclonal; used at 1:2)for 30 min at room temperature. Cells are washed once with 0.1 M PBS, pH7.4. Cells are fixed for 20 min at room temperature with ice-cold 4%paraformaldehyde. Cells are washed twice for 5 min with 0.1 M PBS, pH7.4. Cells preparations are blocked for 30 min at room temperature in10% horse serum (“HS”) diluted in 0.1M PBS, pH 7.4. Cells are incubatedwith secondary antibodies (donkey anti mouse IgG/Alexa488 used at 1:500,(Invitrogen, Cat#A21202); or goat anti mouse IgM/Alexa 488, (Invitrogen,Cat#A21042) diluted in 1% HS for 1 hr at room temperature in the dark.Preparations are washed twice for 5 min with 0.1 M PBS in the dark.Preparations are mounted onto slides face down with mounting medium(Vectashield Mounting Medium, Vector Laboratories, cat#H-1000) or lefton culture wells for quantification and qualification of staining andstored at 4° C.

In some instances stain with Hoechst (a nuclear stain) may be used asfollows. Cells prepared as above are washed with Hoechst solution(diluted 1:10,000 in 0.1% saponin, Sigma, Cat#54521). Cells areincubated in Hoechst solution for 5 min at room temperature, followed by2 washes in 0.1M PBS.

Example 5 Differentiation of OPCs

In a second differentiation protocol, the OPCs are induced todifferentiate by removal of the growth factor mitogens and provision of1% serum. This differentiation protocol produces cell cultures highlyenriched in oligodendrocytes.

In a third differentiation protocol, the OPCs are induced todifferentiate by removal of the growth factor mitogens and provision of30 nM T3 (Sigma, cat#T5516). This differentiation protocol produces cellcultures highly enriched in oligodendrocytes.

Example 6 Encapsulation

If the OPCs are encapsulated, then the following procedure may be used:The hollow fibers are fabricated from a polyether sulfone (PES) with anoutside diameter of 720 m and a wall thickness of a 100 m (AKZO-NobelWuppertal, Germany). These fibers are described in U.S. Pat. Nos.4,976,859 and 4,968,733, herein incorporated by reference. The fiber maybe chosen for its molecular weight cutoff. A PES#5 membrane which has aMWCO of about 280 kd is occasionally used. In other studies, a PES#8membrane which has a MWCO of about 90 kd may be used.

The devices typically comprise: 1) a semipermeable poly(ether sulfone)hollow fiber membrane fabricated by AKZO Nobel Faser AG; 2) a hubmembrane segment; 3) a light cured methacrylate (LCM) resin leading end;and 4) a silicone tether.

The semipermeable membrane used typically has the followingcharacteristics: Internal Diameter 500+30 m Wall Thickness 100+15 mForce at Break 100+15 cN Elongation at Break 44+10% HydraulicPermeability 63+8 (ml/min m² mmHg) nMWCO (dextrans) 280+20 kd.

The components of the device are commercially available. The LCM glue isavailable from Ablestik Laboratories (Newark, Del.); Luxtrak AdhesivesLCM23 and LCM24). The tether material is available from SpecialtySilicone Fabricators (Robles, Calif.). The tether dimensions are 0.79 mmODX0.43 mm IDXlength 202 mm. The morphology of the device is as follows:The inner surface has a permselective skin. The wall has an open cellfoam structure. The outer surface has an open structure, with pores upto 1.5 m occupying 30+5% of the outer surface.

Fiber material is first cut into 5 cm long segments and the distalextremity of each segment sealed with a photopolymerized acrylic glue(LCM-25, ICI). Following sterilization with ethylene oxide andoutgassing, the fiber segments are loaded with a suspension of between10⁴-10⁷ cells, either in a liquid medium, or a hydrogel matrix (e.g., acollagen solution (Zyderm™), alginate, agarose or chitosan) via aHamilton syringe and a 25 gauge needle through an attached injectionport. The proximal end of the capsule is sealed with the same acrylicglue.

A silicone tether (Specialty Silicone Fabrication, Taunton, Ma.) (ID:690 m; OD: 1.25 mm) is placed over the proximal end of the fiberallowing easy manipulation and retrieval of the device.

Example 7 Transplantation of OPCs

Target OPCs may be transplanted into rodent brain to assess graftviability, integration, phenotypic fate of the grafted cells, as well asbehavioral changes associated with grafted cells in healthy animals.

Transplantation is performed according to standard techniques. Forexample, adult rats are anesthetized with sodium pentobarbitol (45mg/kg, i.p.) and positioned in a Kopf stereotaxic instrument. A midlineincision is made in the scalp and a hole drilled for the injection ofcells. Rats receive implants of target OPCs into the striatum using aglass capillary attached to a 10 μl Hamilton syringe. Each animalreceives a total of about 250,000-500,000 cells in a total volume of 2μl. Cells are transplanted 1-2 days after passaging and the cellsuspension is made up of undifferentiated OPC clusters of 5-20 cells.Following implantation, the skin is sutured closed.

Example 8 Transplantation of OPCs into Rodent Models of DysmyelinatingDisease

Target OPCs may be transplanted into rodent brain to assess graftviability, integration, phenotypic fate of the grafted cells, as well asbehavioral changes associated with grafted cells in lesioned or diseasedanimals.

Transplantation is performed according to standard techniques. Forexample, newborn and rats or jimpy mice are anesthetized by hypothermiaand positioned in a Kopf stereotaxic instrument. A midline incision ismade in the scalp and a hole drilled for the injection of cells. Animalsreceive implants of target OPCs into the corpus callosum, fimbria,cerebellar peduncle and/or spinal cord using a glass capillary attachedto a 10 μl Hamilton syringe. Each animal receives a total of about300,000-600,000 cells in a total volume of 6 μl. Cells are transplantedimmediately or 1-2 days after passaging and the cell suspension is madeup of undifferentiated single OPCs or clusters of 5-20 cells. Followingimplantation, the skin is sutured or staple closed.

Alternatively, newborn or juvenile shiverer mice are anesthetized byhypothermia or isofluorane and positioned in a Kopf stereotaxicinstrument. A midline incision is made in the scalp and a hole drilledfor the injection of cells. Mice receive implants of target OPCs intothe corpus callosum, fimbria, cerebellar peduncle and/or spinal cordusing a glass capillary attached to a 10 μl Hamilton syringe. Eachanimal receives a total of about 300,000-600,000 cells in a total volumeof 6 μl. Cells are transplanted immediately or 1-2 days after passagingand the cell suspension is made up of undifferentiated single OPCs orclusters of 5-20 cells. Following implantation, the skin is sutured orstaple closed.

Example 9 Treatment of Neurodegenerative Disease Using Progeny of TargetOPCs In Vitro

Target OPCs are obtained from fetal brain tissue following routinesuction abortion which is collected into a sterile collection apparatus.A 2×4×1 mm piece of tissue is dissected and dissociated as in Examples 1or 2. Target OPCs are then proliferated. The target OPC progeny are usedfor neurotransplantation into a blood-group matched host with aneurodegenerative disease. Surgery is performed using a BRW computedtomographic (CT) stereotaxic guide. The patient is given localanesthesia suppiemencea with intravenously administered midazolam. Thepatient undergoes CT scanning to establish the coordinates of the regionto receive the transplant. The injection cannula consists of a 17-gaugestainless steel outer cannula with a 19-gauge inner stylet. This isinserted into the brain to the correct coordinates, then removed andreplaced with a 19-gauge infusion cannula that has been preloaded with30 μl of tissue suspension. The cells are slowly infused at a rate ofabout 3 μl/min as the cannula is withdrawn. Multiple stereotactic needlepasses are made throughout the area of interest, approximately 4 mmapart. The patient is examined by CT scan postoperatively for hemorrhageor edema. Neurological evaluations are performed at variouspost-operative intervals, as well as PET scans to determine metabolicactivity of the implanted cells.

Example 10 Genetic Modification of Target OPC Progeny Using CalciumPhosphate Transfection

Target OPC progeny are propagated as described herein. The cells arethen transfected using a calcium phosphate transfection technique. Forstandard calcium phosphate transfection, the cells are mechanicallydissociated into a single cell suspension and plated on tissueculture-treated dishes at 50% confluence (50,000-75,000 cells/cm²) andallowed to attach overnight.

The modified calcium phosphate transfection procedure is performed asfollows: DNA (15-25 μg) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA,pH 7.5) diluted to 440 μl with TE, and 60 μl of 2M CaCl₂ (pH to 5.8 with1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 μl of 2×HeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na₂ HPO₄, 12mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is added dropwise tothis mix. The mixture is allowed to stand at room temperature for 20minutes. The cells are washed briefly with 1× HeBS and 1 ml of thecalcium phosphate precipitated DNA solution is added to each plate, andthe cells are incubated at 37° for 20 minutes. Following thisincubation, 10 mls of complete medium is added to the cells, and theplates are placed in an incubator (37° C., 9.5% CO₂) for an additional3-6 hours. The DNA and the medium are removed by aspiration at the endof the incubation period, and the cells are washed 3 times with completegrowth medium and then returned to the incubator.

Example 11 Genetic Modification of Target OPCs Using Viral Vectors

Target OPCs are proliferated as described herein and then infected withlentiviral vectors containing genes of interest in addition to a reportgene such as GFP (green fluorescence protein). Lentivirus suspensionsare added to the culture medium where OPCs are proliferated andincubated for 24 hours. After 24 h, the culture medium is removed andreplaced with fresh medium and the OPCs are cultured for another 3 days.Cells are collected and centrifuged and cells expressing the gene ofinterest are sorted by flow cytometry. Positive cells are returned tothe proliferative medium.

The transduced OPC progeny are transplanted into a rodent or humanpatient using the procedures described in the previous Examples.

Example 12 Transplantation of OPCs into Rodent Models of Spinal CordInjury

Target OPCs may be transplanted into rodent spinal cord to assess graftviability, integration, phenotypic fate of the grafted cells, as well asbehavioral changes associated with grafted cells in spinal cord lesionedanimals.

Animals receive a laminectomy at vertebral level T9. Animals thenreceive a 50-kilodyne (kd) contusion spinal cord injury using anInfinite Horizon Impactor (Precision Systems and Instrumentation,Lexington, Ky.). Seven days after spinal cord injury, mice are testedusing the Basso, Beattie, and Bresnahan (BBB) rating scale andrandomized to receive OPCs or vehicle control. Cells are injectedbilaterally anterior and posterior to the epicenter of the lesion usinga beveled grass micropipette affixed to a nanoinjector device. Eachanimal receives 50,000 to 80,000 cells.

Example 13 Transplantation of OPCs into Rodent Models of MS

Myelin oligodendrocyte glycoprotein (MOG)-induced murine experimentalautoimmune encephalomyelitis (EAE) is a widely accepted model forstudying the clinical and pathological features of multiple sclerosis.

Transplantation is performed according to standard techniques. Forexample, adult animals affected by MOG-induced EAE are anesthetizedusing isoflurane gas and positioned in a Kopf stereotaxic instrument. Amidline incision is made in the scalp and a hole drilled for theinjection of cells. Animals receive implants of target OPCs into thecorpus callosum, fimbria, cerebellar peduncle, lateral ventricular spaceand/or spinal cord using a glass capillary attached to a 10 μl Hamiltonsyringe. Each animal receives a total of about 300,000-600,000 cells ina total volume of 6 μl. Cells are transplanted immediately or 1-2 daysafter passaging and the cell suspension is made up of undifferentiatedsingle OPCs or clusters of 5-20 cells. Following implantation, the skinis sutured or staple closed.

Example 14 Characterization of the Engraftment Ability of ExpandedPDGFR⁺CD105⁻ Oligodendrocyte Progenitor (OPC) Population

In order to determine whether FACS-isolated, in vitro expandedoligodendrocyte progenitor cells survive, migrate and are capable of invivo myelination, a series of transplantation studies were conductedusing the shiverer mouse, a rodent model of dysmyelination. The shiverermouse contains a naturally occurring deletion of a large portion of thembp gene which results in incomplete CNS myelin formation. In order toavoid xenogenic rejection of human cells, the shiverer mice werebackcrossed to the immunodeficient NOD-Scid mouse. Engraftment ofoligodendrocyte progenitors was studied in two different Shiverer/Scidage groups: as juveniles (P21-P30) and as neonates (P0-P1).Shiverer/Scid mice have a relatively short lifespan of around 8 weeksand therefore the longest post transplantation time point studied wasabout 8 weeks in the case of neonatal injections and 5 weeks forjuvenile injections.

Oligodendrocyte progenitors are grown as a monolayer. In order toprepare cells for transplantation, OPCs were lifted off the flasks usingtrypsin, following a protocol similar to that used to passage OPCs.Cells were then exposed to trypsin inhibitor to stop the proteolyticdigestion, washed twice in culture medium and ressuspended in ex-vivomedium containing the antioxidant NAC (1 mM) at a final density of 1E5cells/μl.

The ability for OPCs to be held in a suspension culture format for atleast one day post trypsin treatment was tested. This 1-day hold periodin suspension could be advantageous for 3 reasons: 1) would allow cellsto recover from a potentially damaging enzyme treatment prior totransplantation and/or cryopreservation; 2) it would introduce moreflexibility in animal surgery scheduling and 3) would make shipment ofready-to-transplant OPCs to an off-site location (laboratory or clinic)possible. Finally, and given the potential application of OPCs forclinical application, the engraftment ability and myelination potentialof OPCs after cryopreservation was tested with positive results.

Example 15 Transplantation

Juvenile or neonatal shiverer/Scid mice were placed in a stereotaxicframe and 1 μl of OPC suspension was injected into 2-3 brain locations,bilaterally (4-6 total injections/mouse). Injections targeted the corpuscallosum, fimbria and the cerebellar peduncle (see FIG. 1), regions ofthe brain that are heavily myelinated (rich in white matter) inmyelin-competent animals but severely hypomyelinated in theshiverer/scid mouse. Mice were sacrificed at different time points, upto 8 weeks, and their brains analyzed for the presence of human cellsusing the monoclonal antibody SC121 and for the presence of humanderived oligodendrocytes, capable of myelinating mouse axons, using anMBP antibody. Because shiverer mice have a mutation that deletes most ofthe mbp gene, MBP protein is not produced by mouse oligodendrocytes andtherefore any MBP detected by the anti-MBP antibody is of human origin.

TABLE 15-1 Summary of engraftment data obtained from 4 OPC lines derivedfrom 4 different donor tissues. Total # Presence Myelin Shiverer agetransplanted of donor production Donor ID, age Passage tested groupShi/Scid mice cells SC121⁺ MBP⁺ 2657, 18 wks 3, 13 & 14 Juvenile 1010/10 10/10 2703, 18 wks 6, 9, 14, 15 & 16 Juvenile & 4 juv + 15 neo19/19 19/19 Neonates 2710, 18 wks 2 Neonates 2 2/2 2/2 2711, 18 wks 4,6, 11 & 12 Neonates 16 16/16 16/16

All juvenile and neonatal animals injected with OPCs contained humancells (as determined by SC 121 staining) at all ages tested. Whenstained for MBP, all animals also demonstrated positive staining (seeFIG. 2 for one example of SC121 and MBP staining in serial sections).

The possibility of genetically modifying OPCs with lentiviral vectorswas also tested. As a proof of concept, we used a lentiviral vectorexpressing the reporter gene GFP (green fluorescence protein) under thecontrol of the MBP promoter. This allows for direct visualization(antibody staining free) of the OPCs that are actively transcribing theMBP gene (bright green cells) and that have the morphology of maturemyelinating oligodendrocytes (multiple processes with cable likemorphology, aligned with axonal bundles). FIG. 3 shows one example of ashiverer/scid mouse transplanted with MBP-GFP OPCs, sacrificed at 8weeks post transplantation.

The in vivo study showed that neonates and juvenile shiverer mice areappropriate models for testing engraftment and myelination ability ofexpanded OPCs. Major differences in the engraftment quality betweenneonate and juvenile shiverer/scid mice were not observed.

The in vivo study showed that engraftment of OPCs and myelination werenot significantly affected by passage number; for instance, in neonatesthat were injected with donor 2703, there was no qualitative differencein the total number of human cells and the extent of myelination,suggesting that potency (ability to engraft and to myelinate) does notdiminish with passage up to passage 16, the longest passage numbertested.

The in vivo study showed that when the potency of freshly passaged(never frozen) OPCs is compared with that of previously cryopreservedOPCs at the same or similar passage, there were no major qualitativedifferences in engraftment and myelination. Although only neonatalanimals were transplanted with previously cryopreserved cells, we do notexpect a different outcome in juvenile animals. This result indicatesthat OPC cultures can be cryoprotected without any detectable loss inpotency. Similarly, there is no loss of potency (engraftment ormyelination ability) due to the one-day hold period, suggesting thatthis protocol can be used to facilitate OPC culture transfers to offsite locations in a ready-to-use format.

EQUIVALENTS

The details of one or more embodiments of the invention are set forth inthe accompanying description above. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this

1. An enriched population of oligodendrocyte precursor cells (OPCs)derived from neural or neural derived cells, wherein the population isenriched for target OPCs that are PDGFRα⁺ and CD105⁻, wherein at least30% of the cells in the population are target OPCs.
 2. The population ofclaim 1, where in the target cells are also PSA-NCAM^(lo/−).
 3. Thepopulation of claim 1, where in the target cells are also A2B5^(lo/−).4. The population of claim 1, where in the target cells are also CD133⁺.5. The population of OPCs in claim 1, wherein at least 50% of the cellsin the population are target OPCs.
 6. The population of OPCs in claim 1,wherein at least 70% of the cells in the population are target OPCs. 7.The population of OPCs in claim 1, wherein at least 90% of the cells inthe population are target OPCs.
 8. The population of OPCs in claim 1,wherein the target OPCs are PDGFRα⁺, CD105⁻, CD133⁺, and A2B5⁻.
 9. Thepopulation of OPCs in claim 1, wherein the target OPCs are PDGFRα⁺,CD105⁻, CD133⁺, A2B5^(lo/−), and PSA-NCAM⁻.
 10. A method of producingpopulation of cells enriched for target oligodendrocyte precursor cells(OPCs) from neural or neural derived cells, comprising contacting neuralor neural derived cells with at least one reagent that binds to cellsurface antigens expressed by the target OPCs, wherein the target OPCsare PDGFRα⁺ and CD105⁻, wherein the population of cells is enriched tocontain at least 30% target OPC.
 11. The method of claim 10, wherein thetarget OPCs are also PSA-NCAM^(lo/−).
 12. The method of claim 10, wherein the target cells are also A2B5^(lo/−).
 13. The method of claim 10,where in the target cells are also CD133⁺.
 14. The method of claim 10further comprising negatively selecting for target OPCs by selectivelyremoving nontarget cell populations.
 15. The method of claim 10, whereinthe target OPCs are PDGFRα⁺, CD105⁻, CD133⁺, and A2B5⁻.
 16. The methodof claim 10, wherein the target OPCs are PDGFRα⁺, CD105⁻, CD133⁺,A2B5^(lo/−), and PSA-NCAM⁻.
 17. A method of increasing the number oftarget oligodendrocyte precursor cells (OPCs) in a cell culturecomprising culturing a population of cells comprising at least 30%target OPCs to increase the number of target OPCs in the cell culture,wherein the target OPCs are PDGFRα⁺ and CD105⁻.
 18. The method of claim17, wherein the target OPCs are also PSA-NCAM^(lo/−).
 19. The method ofclaim 17, where in the target cells are also A2B5^(lo/−).
 20. The methodof claim 17, where in the target cells are also CD133⁺.
 21. The methodof claim 17, wherein the target OPCs are PDGFRα⁺, CD105⁻, CD133⁺, andA2B5⁻.
 22. The method of claim 17, wherein the target OPCs are PDGFRα⁺,CD105⁻, CD133⁺, A2B5^(lo/−), and PSA-NCAM⁻.
 23. The method of claim 17further comprising contacting the OPCs with an effective amount of atleast one biological agent that is capable of increasing the number ofOPCs.
 24. The method of claim 23 wherein the biological agent isselected from the group consisting of leukemia inhibitory factor (LIF),epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2;bFGF), IGF-1, NT3, Shh, CTNF, PDGF-AA and combinations thereof.
 25. Amethod for proliferating oligodendrocyte precursor cells (OPCs)comprising: proliferating OPCs in serum-free culture medium containingone or more predetermined growth factors effective for inducing OPCproliferation, wherein: (a) the population comprises OPCs which arePDGFRα⁺ and CD105⁻; and (b) in the presence of differentiation-inducingconditions, the cells produce progeny cells that differentiate intooligodendrocytes.
 26. The method of claim 25, wherein the OPCs are alsoPSA-NCAM^(lo/−).
 27. The method of claim 25, where in the target cellsare also A2B5^(lo/−).
 28. The method of claim 25, where in the targetcells are also CD133⁺.
 29. The method of claim 25 further comprisingsubjecting the OPCs to culture conditions that induce oligodendrocytedifferentiation to produce differentiating and differentiatedoligodendrocytes.
 30. A composition comprising the oligodendrocyteprecursor cells (OPCs) produced by the method of claim
 25. 31. Acomposition comprising the differentiating or differentiatedoligodendrocytes produced by the method of claim
 25. 32. A method oftreating a mammalian individual suffering from a disease associated withdemyelination of central nervous system axons, comprising introducingthe enriched population of OPCs of claim 1 to the mammalian individualin an amount effective to treat the disease.
 33. The method of claim 32,wherein the mammalian individual is a human.
 34. The method of claim 32,wherein the OPCs are administered to the mammalian individual by celltransplantation.
 35. The method of claim 32, wherein the disease ismultiple sclerosis, Pelizaeus-Merzbacher disease, cerebral palsy,radiation induced myelination disorders, acute disseminatedencephalomyelitis, transverse myelitis, demyelinating genetic disease,spinal cord injury, virus-induced demyelination, Progressive MultifocalLeucoencephalopathy, Human Lymphotrophic T-cell Virus I(HTLVI)-associated myelopathy, or nutritional metabolic disorder. 36.The method of claim 32, wherein the disease is multiple sclerosis,Pelizaeus-Merzbacher disease, or cerebral palsy.
 37. A method oftreating a mammalian individual suffering from a disease associated withdemyelination of central nervous system axons, comprising introducingthe OPCs of claim 1 to the mammalian individual in an amount effectiveto treat the disease.
 38. The method of claim 37, wherein the mammalianindividual is a human.
 39. The method of claim 37, wherein the OPCs areadministered to the mammalian individual by cell transplantation. 40.The method of claim 37, wherein the disease is multiple sclerosis, acutedisseminated encephalomyelitis, transverse myelitis, demyelinatinggenetic disease, spinal cord injury, virus-induced demyelination,Progressive Multifocal Leucoencephalopathy, Human Lymphotrophic T-cellVirus I (HTLVI)-associated myelopathy, or nutritional metabolicdisorder.
 41. A method of treating a mammalian individual suffering froma disease associated with demyelination of central nervous system axons,comprising introducing the differentiating or differentiatedoligodendrocytes prepared according to the method of claim 21 to themammalian individual in an amount effective to treat the disease. 42.The method of claim 41, wherein the mammalian individual is a human. 43.The method of claim 41, wherein the differentiating or differentiatedoligodendrocytes are administered to the mammalian individual by celltransplantation.
 44. The method of claim 41, wherein the disease ismultiple sclerosis, acute disseminated encephalomyelitis, transversemyelitis, demyelinating genetic disease, spinal cord injury,virus-induced demyelination, Progressive Multifocal Leucoencephalopathy,Human Lymphotrophic T-cell Virus I (HTLVI)-associated myelopathy, ornutritional metabolic disorder.
 45. A method of screening for compoundsthat affect a biological function of an enriched population of targetoligodendrocyte precursor cells comprising: (a) contacting an enrichedpopulation of target oligodendrocyte precursor cells obtained by themethod of claim 1 with a test compound; and (b) detecting a change in abiological function of the oligodendrocyte precursor cells.
 46. Themethod of claim 45, wherein said target oligodendrocyte precursor cellsare PDGFRα⁺, CD105⁻, A2B5^(lo/−), PSA-NCAM^(lo/−), and CD133⁺.
 47. Themethod of claim 45, wherein said target oligodendrocyte precursor cellsare PDGFRα⁺, CD105⁻, A2B5⁻, and CD133⁺.
 48. The method of claim 45,wherein said target oligodendrocyte precursor cells are PDGFRα⁺, CD105⁻,A2B5^(lo/−), PSA-NCAM⁻, and CD133⁺.
 49. The method of claim 45, whereinthe change occurs in at least one of the characteristics selected fromthe group consisting of myelination, differentiation intooligodendrocytes, proliferation rate, cell migration, viability, geneexpression, protein expression, protein levels in the culturing medium,dedifferentiation, growth characteristics, and cell morphology.
 50. Amethod of producing a population enriched for oligodendrocyte precursorcells comprising: (a) contacting neural or neural derived cellscomprising one or more multipotent central nervous system stem cell withan antibody that specifically binds to PDGFRα; and (b) selecting saidneural or neural derived cells that are PDGFRα^(hi), wherein theselected cells are enriched for oligodendrocyte precursor cells ascompared with the neural or neural derived cells.
 51. The method ofclaim 50, wherein said neural or neural derived cells are obtained froma neuro sphere culture or an adherent culture.
 52. The method of claim50, wherein the method further comprises the step of eliminating thosecells that are PDGFRα^(lo/med).
 53. The method of claim 50, wherein themethod further comprises the step of eliminating cells that are CD105⁻.